Project Summary: ?Single-cell magnetic microscopy with multicolor superparamagnetic probes. The ability to image small tumors or individual circulating tumor cells non-invasively could have a major impact on the early detection of cancer. However, this remains a major technical challenge, as target cells must be abundantly labeled with contrast agents with a high degree of specificity, and ultra-sensitive imaging modalities must be employed that retain sensitivity even when separated by thick tissue. Superparamagnetic iron-oxide nanoparticles (SPIONs) are promising probes for these purposes, as they are relatively non-toxic and magnetic fields are not attenuated by tissue. Our group has recently developed a technique, diamond magnetic microscopy, capable of imaging individual 22-nm SPIONs within ~1 m of the diamond surface with high spatial resolution (~300 nm), wide field of view (up to several mm), and >1 kHz frame rate. Here we propose a research program to extend the platform to image immunomagnetically labeled cells using multicolor SPION probes and diamond magnetic microscopy. Individual ?colors? of SPIONs will be sorted based on their relaxation times using a microfluidic chamber outfitted with miniature magnets. We will use these probes in combination with diamond magnetic microscopy for two proof-of-principle experiments. In the first, two SPION colors will be introduced to breast cancer cells ?in vitro?; one batch will be functionalized with the Her2 antibody and the other will be labeled with isotype-matched non-specific antibody. The cells will be fixed at various timesteps afterwards and imaged using a dual-channel diamond magnetic microscope. The microscope can differentiate SPION ?colors? based on their different temporal response to pulsed magnetic fields, revealing images of the sub-cellular distribution of specific and non-specifically-bound SPIONs. These data will be used to unravel the evolution of specific and non-specific internalization dynamics. In the second experiment, we will use a scaled-up version of the diamond magnetic microscope to detect individual magnetically-labeled cells flowing at physiological rates (up to 1 cm/s) through a 1-mm-thick tissue phantom. To accomplish these aims, we have assembled a team with broad strengths, including SPION chemistry, cancer biology, and sensor physics. If successful, the detection platform may eventually be used to detect CTCs and small tumors ?in vivo?.